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beta-D-Xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2). Biphasic inhibition by triethanolamine of E186A preparations reveals minor contamination by wild-type-like enzyme, the contaminant likely originating from translational misreading. Titration of E186A preparations with triethanolamine allows resolution of binding and kinetic parameters of the E186A mutant from those of the contaminant. The E186A mutation abolishes the pKa assigned to E186; mutant enzyme binds only the neutral aminoalcohol pH-independent K(triethanolamine)(i)=19 mM), whereas wild-type enzyme binds only the cationic aminoalcohol pH-independent K(triethanolamine)(i)=0.065 mM. At pH 7.0 and 25 degrees C, relative kinetic parameter, k(4NPX)(cat)=k(4NPA)(cat), for substrates 4-nitrophenyl-beta-D-xylopyranoside (4NPX) and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA) of E186A is 100-fold that of wild-type enzyme, consistent with the view that, on the enzyme, protonation is of greater importance to the transition state of 4NPA whereas ring deformation dominates the transition state of 4NPX.

Beta-D-Xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D: -xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), and 1,4-beta-D-xylobiose (X2) was determined on and off (k (non)) the enzyme at pH 5.3, which lies in the pH-independent region for k (cat) and k (non). Rate enhancements (k (cat)/k (non)) for 4NPX, 4NPA, and X2 are 4.3 x 10(11), 2.4 x 10(9), and 3.7 x 10(12), respectively, at 25 degrees C and increase with decreasing temperature. Relative parameters k (cat) (4NPX)/k (cat) (4NPA), k (cat) (4NPX)/k (cat) (X2), and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(X2) increase and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(4NPA), (1/K (m))(4NPX)/(1/K (m))(4NPA), and (1/K (m))(4NPX)/(1/K (m))(X2) decrease with increasing temperature.

Catalytically efficient beta-D-xylosidase from Selenomonas ruminantium (SXA) exhibits pK (a)s 5 and 7 (assigned to catalytic base, D14, and catalytic acid, E186) for k (cat)/K (m) with substrates 1,4-beta-D-xylobiose (X2) and 1,4-beta-D-xylotriose (X3). Catalytically inactive, dianionic SXA (D14(-)E186(-)) has threefold lower affinity than catalytically active, monoanionic SXA (D14(-)E186(H)) for X2 and X3, whereas D14(-)E186(-) has twofold higher affinity than D14(-)E186(H) for 4-nitrophenyl-beta-D-xylopyranoside (4NPX), and D14(-)E186(-) has no affinity for 4-nitrophenyl-alpha-L-arabinofuranoside. Anomeric isomers, alpha-D-xylose and beta-D-xylose, have similar affinity for SXA. 4-Nitrophenol competitively inhibits SXA-catalyzed hydrolysis of 4NPX. SXA steady-state kinetic parameters account for complete progress curves of SXA-catalyzed hydrolysis reactions.

Beta-D-xylosidase from the ruminal anaerobic bacterium, Selenomonas ruminantium (SXA), catalyzes hydrolysis of beta-1,4-xylooligosacharides and has potential utility in saccharification processes. The enzyme, heterologously produced in Escherichia coli and purified to homogeneity, has an isoelectric point of approx 4.4, an intact N terminus, and a Stokes radius that defines a homotetramer. SXA denatures between pH 4.0 and 4.3 at 25 degrees C and between 50 and 60 degrees C at pH 5.3. Following heat or acid treatment, partially inactivated SXA exhibits lower k(cat) values, but similar K(m) values as untreated SXA. D-glucose and D-xylose protect SXA from inactivation at high temperature and low pH.

Beta-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl- beta-D-xylopyranoside (4NPX) and 1,4-beta-D-xylobiose (X2). Progress curves of 4-nitrophenyl-beta-D-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of kcat and kcat/Km decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites -1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond -1 are blocked by protein and subsites beyond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode kcat and kcat/Km by <103 and to a similar extent for substrates 4NPX and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs kcat/Km with pKa values of 5.0 and 7.0 assigned to D14 and E186, respectively. kcat(4NPX) has a pKa value of 7.0 and kcat(4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, "dianionic" enzyme form (D14-E187-) binds 4NPX but not 4NPA.